The present invention relates to methods for the separation, in particular, the preparative separation of amphoteric macromolecules by two-dimensional electrophoresis, whereby isoelectric focusing in liquid media is combined with electrophoresis, and subsequent electroelution to liquid media. The invention further relates to a system for separation of amphoteric macromolecules, comprising an isoelectric focusing unit, a composite body having channels accommodating the electrophoresis medium and an elution plate for use in said methods.
The principles for two-dimensional electrophoresis are known in the art. This technique has normally involved first subjecting a mixture of molecules to isoelectric focusing on a gel where a pH gradient has been established. As the molecules traverses the pH gradient they reach at some point a pH corresponding to their respective isoelectric point and stop migrating. Traditionally, this separation has been performed in a capillary gel, which then has been placed on top of a slab gel in order to subject the sample to SDS-PAGE, whereby the molecules are separated according to their molecular weights. In order to further process the separated molecules, additional handling steps are required, for example detecting the spots, cutting the gel pieces out of the slab gel and extraction of the material from the gel. A problem with this method is low recovery rates. Another problem with this method is that it is a largely manual procedure which requires skill to perform it and which is time-consuming to perform.
Isoelectric focusing can also be performed in liquid media, as disclosed in U.S. Pat. No. 4,971,670. A multi-chambered isoelectric focusing unit (IsoPrime(trademark) unit) is commercially available. With this unit, a pH-graduated set of buffered polyacrylamide membranes separates a series of chambers through which a sample circulates. Each membrane defines a specified step in a pH gradient. Each chamber encompasses a pH (or pI) range determined by the pH values of the membranes that bound it. During electrophoresis, a protein passes through membranes into successive chambers until it reaches the chamber encompassing its pI. The protein remains focused in this chamber and can subsequently be recovered from the chamber. This process is intended to separate and purify one amphoteric compound from a flow of compound. It is not suitable for separating a plurality of compounds that are to be analysed and which have the same isoelectric point as all compounds having the same isoelectric point end up in the same chamber. They then have to be extracted from the chamber and further separated by some other means.
A composite body having channels suitable for accommodating gels for electrophoresis is disclosed in WO 97/37216. Said composite body comprises a wall structure made from integrally formed element having a plurality of parallel longitudinally extending channels, said channels accommodating chemical medium or media suitable for carrying out a test, analysis or reaction procedure in situ in the channels.
However, the presently known methods for separating mixtures of amphoteric macromolecules often requires multiple manual steps and are difficult to automate. In addition, the known methods are not optimised to prepare samples suitable for direct use in downstream applications, such as e.g. mass spectrometry.
The present invention provides a method for separating mixtures of amphoteric macromolecules which avoids the above mentioned drawbacks and is designed for automation. With the use of the methods according to the invention, samples are obtained in solution, ready for downstream processing by means of e.g. mass spectrometry or other characterisation methods.
Consequently, in a first aspect the invention provides a method for separating amphoteric macromolecules, e.g. proteins or peptides, comprising the steps
(a) subjecting said mixture of macromolecules to isoelectric focusing in liquid media;
(b) collecting samples from step (a), said samples containing macromolecules separated on basis of isoelectric point, and transferring each sample to a channel accommodating medium for electrophoresis, said channel being part of a composite body;
(c) subjecting said samples, contained in the channels in said composite body, to electrophoresis; and
(d) allowing electrophoresis to proceed until macromolecules are eluted from said medium for electrophoresis, and collecting fractions of the samples containing macromolecules.